The health of the oral cavity is maintained by salivary secretions. The principal function of salivary glands is to produce these complex fluids. We utilize in vitro dispersed cells, and cultured epithelial cells of salivary glands, to understand mechanisms controlling saliva formation. We have focused our studies on neurotransmitter regulation of secretory events and associated signalling mechanisms. During this reporting period the primary focus of study has been muscarinic receptors in rat parotid gland acinar cells and their coupling to functional responses via specific G proteins. In these cells, stimulation of muscarinic receptors results in the generation of inositol phosphates via the activation of a phosphatidylinositol 4,5- bisphosphate specific phospholipase C. Subsequently this response leads to the elevation of cytosolic Ca2+ levels and fluid secretion. Additionally, muscarinic receptors can mediate the inhibition of agonist induced cAMP formation. We have examined the binding of a subtype non-selective antagonist (quinuclidinyl benzilate, QNB) to parotid muscarinic receptors in vitro and in vivo. In vitro, [3H] QNB binds to an homogenous population of receptors (Bmax about 550 fmol/mg protein) with high affinity (Kd about 100 pM). In vivo, we have employed a pharmacokinetic approach, with stereospecific iodinated QNB enantiomers. Acinar cells contain a single pharmacologically detectable subtype of muscarinic receptor, the M3, which appears to regulate both measured functional responses. Additionally, by Northern blot analyses, mRNA for only the M3 receptor subtype is detected, while immunochemical studies show that M3 receptors account for 93% of all immuno-precipitable receptors in these cells.